Rumen

We report benchmark results for two additional datasets, consisting of sequencing reads from Illumina HiSeq 4000 paired end sequencing (2 × 150 base pairs) and Illumina HiSeq 2500 paired end sequencing (2 × 250 base pairs). The datasets were created based on data from a recent rumen metagenome study^{31 (link)} (Supplementary Information, see Supplementary Benchmark 1) and an environmental study of the topsoil microbiome³² (Supplementary Information, see Supplementary Benchmark 2). SCOPe-annotated datasets of 1.55 million and 1 million reads, respectively, were obtained as described in the Supplementary Information. The benchmark runs for the two query read datasets were carried out analogously to the run for our main benchmark, operating all tools in translated search mode against the same database of SCOPe-annotated UniRef50 sequences. We report performance, AUC1 values and ROC curves for both runs (Extended Data Figs. 4–7).

The Iso-Seq method for sequencing full-length transcripts was developed by PacBio during the same time period as the genome assembly. We therefore used this technique to improve characterization of transcript isoforms expressed in cattle tissues using a diverse set of tissues collected from L1 Dominette 0 1449 upon euthanasia. The data were collected using an early version of the Iso-Seq library protocol [26 ] as suggested by PacBio. Briefly, RNA was extracted from each tissue using Trizol reagent as directed (Thermo Fisher). Then 2 μg of RNA were selected for PolyA tails and converted into complementary DNA (cDNA) using the SMARTer PCR cDNA Synthesis Kit (Clontech). The cDNA was amplified in bulk with 12–14 rounds of PCR in 8 separate reactions, then pooled and size-selected into 1–2, 2–3, and 3–6 kb fractions using the BluePippin instrument (Sage Science). Each size fraction was separately re-amplified in 8 additional reactions of 11 PCR cycles. The products for each size fraction amplification were pooled and purified using AMPure PB beads (Pacific Biosciences) as directed, and converted to SMRTbell libraries using the Template Prep Kit v1.0 (PacBio) as directed. Iso-Seq was conducted for 22 tissues including abomasum, aorta, atrium, cerebral cortex, duodenum, hypothalamus, jejunum, liver, longissimus dorsi muscle, lung, lymph node, mammary gland, medulla oblongata, omasum, reticulum, rumen, subcutaneous fat, temporal cortex, thalamus, uterine myometrium, and ventricle from the reference cow, as well as the testis of her sire. The size fractions were sequenced in either 4 (for the smaller 2 fractions) or 5 (for the largest fraction) SMRTcells on the RS II instrument. Isoforms were identified using the Cupcake ToFU pipeline [27 ] without using a reference genome.
Short-read–based RNA-seq data derived from tissues of Dominette were available in the GenBank database because her tissues have been a freely distributed resource for the research community. To complement and extend these data and to ensure that the tissues used for Iso-Seq were also represented by RNA-seq data for quantitative analysis and confirmation of isoforms observed in Iso-Seq, we generated additional data, avoiding overlap with existing public data. Specifically, the TruSeq stranded mRNA LT kit (Illumina, Inc.) was used as directed to create RNA-seq libraries, which were sequenced to ≥30 million reads for each tissue sample. The Dominette tissues that were sequenced in this study include abomasum, anterior pituitary, aorta, atrium, bone marrow, cerebellum, duodenum, frontal cortex, hypothalamus, KPH fat (internal organ fat taken from the covering on the kidney capsule), lung, lymph node, mammary gland (lactating), medulla oblongata, nasal mucosa, omasum, reticulum, rumen, subcutaneous fat, temporal cortex, thalamus, uterine myometrium, and ventricle. RNA-seq libraries were also sequenced from the testis of her sire. All public datasets, and the newly sequenced RNA-seq and Iso-Seq datasets, were used to annotate the assembly, to improve the representation of low-abundance and tissue-specific transcripts, and to properly annotate potential tissue-specific isoforms of each gene.

DNA was extracted from the rumen contents using Soil DNA Kit (MOBIO, Carlsbad, CA, USA). The concentration and purity of the extracted DNA were determined with TBS-380 fluorometer (Turner Biosystems, Sunnyvale, CA, USA) and NanoDrop 2000 (NanoDrop Technologies, Wilmington, DE, USA), respectively. The DNA was fragmented to fragments of approximately 400 bp using Covaris M220 (Gene Company Limited, Hongkong, China) for library construction. Sequencing was performed using the Illumina NovaSeq6000 (Illumina Inc, San Diego, CA, USA) sequencing platform. Sequencing adapters were removed with FAST software (version 0.20.0), and low-quality reads (length < 50 bp in length or quality value < 20 or with N bases) were removed [26 (link)]. Reads were aligned to the Bos Taurus reference genome assembly using BWA 0.7.9a (http://bio-bwa.sourceforge.net). Metagenomic sequencing data were assembled with MULTIPLE MEGAHIT (Version 1.1.2) [27 (link)]. Overlapping sequences with lengths ≥ 300 bp were selected as the final assembly results and used for further gene annotation. Metagenes were used to predict the best candidate open reading frames (ORFs) [28 (link)]. The predicted ORFs with length ≥ 100 bp were retrieved. A cluster analysis of a non-redundant gene catalog with sequence homology and 90% coverage was constructed using CD-HIT (version 4.6.1) [29 (link)]. Subsequently, representative sequences of the non-redundant gene catalog were aligned with the NCBI NR database using BLASTP (version 2.2.28 +) (the e-value cutoff for the best match was 1e −5) to obtain annotation results and species abundance degree [30 (link)]. Finally, a BLAST search (version 2.2.28 +) with an optimization criterion cutoff of 1e −5, annotated against the KEGG database [31 (link)], was performed using USEARCH (http://www.drive5.com/usearch/) CAZY annotation [32 ].

On the 270^th d, all experimental animals fasted for 12 h and were weighed. Next then the 18 animals were transported, without mixing treatment groups, to a commercial abattoir and slaughtered within 1 h of their arrival by bolt stunning followed by exsanguination from the jugular vein. Following slaughter, the carcass weight and net meat percentage were measured and calculated according to previously reported methods [24 (link), 25 (link)]. A rumen tissue specimen of approximately 2 cm × 2 cm was immediately removed from the left dorsal sac, fixed in glutaraldehyde solution, and stored at 4 °C for examination under scanning electron microscopy (SEM). Then, 5 mL of the liquid and solid mixture was collected from the left dorsal sac of each test animal, transferred to sterile tubes, and immediately frozen with liquid nitrogen. Six randomly selected samples from each group were sent back to the laboratory the same day and stored at −80 °C for metagenomic and metabolomic sequencing. A further 15 mL of rumen content (18 bulls) was collected and stored in a sterilized container at −20 °C for measurement of digestive enzymes and fermentation parameters.

The rumen contents and homogenate mixture were placed in an ultrasonic beater to obtain a 10% homogenization buffer. The supernatant was collected, and subsequent processed was done in accordance with the kit's (Biosino Biotechnology Co., Ltd., Beijing, China) instructions. The enzyme activities (Lipase, Cellulase, Protease, Xylanase and β-glucosidase) were determined by colorimetry assays [5 ].